The estrogen receptor (ER) and progesterone receptor (PR) content of primary breast carcinomas is well-established as an important factor in predicting the patient's disease-free survival and overall survival. Until recently the receptor content of breast tumors was determined by ligand-binding assays which require substantial amounts of tissue, are difficult, lengthy and expensive to perform. The production of monoclonal antibodies to the human estrogen receptor and, more recently, human progesterone receptor has made more advantageous, immune- based assays possible. We plan to use these antibodies, especially monoclonal PR antibodies, to characterize the receptor content of breast cancers with immunohistochemical assays and determine the utility of these assays in predicting patient response to endocrine manipulative management and in evaluating archival material. One of the short-comings of immunohistochemistry, in general, is the lack of quantitative information derived from these techniques. We will use ER and PR immunohistochemical assays as a prototype for applying computerized image analysis to the quantitation of immunolocalized nuclear proteins. Immunoelectron microscopy and liquid phase immunoprecipitation studies of human cultured breast carcinoma cells under various conditions of hormonal stimulation will be used to characterize the subcellular sites of hormone receptor binding. Complementary DNA (cDNA) coding for ER and PR will be used to determine the chromosomal sites of these genes and to further characterize functional domains. Northern and Southern blot analyses will determine the level at which steroid hormone receptor expression is interrupted in ER- and PR-poor breast cancers. Southern blot analysis will also be used to determine if a proportion of breast cancers have amplification or rearrangement of ER and/or PR genes. A less favorable long-term survival and disease-free interval in breast cancer patients has recently been associated with amplification of the HER-2 (or neu) oncogene, a putative growth factor receptor gene. We, in collaboration with Dr. D.J. Slamon, propose an investigation of HER-2 gene expression comparing production of the protein product with gene amplification and patient survival. Successful completion of this work will provide new methods for characterizing ER and PR and insights into potential mechanisms of hormonal regulation of breast cancer.